Methods of Protein Separation

289 Pages·1975·9.022 MB·English

by  John R. Cann (auth.), Nicholas Catsimpoolas (eds.)

METHODSOF
PROTEIN
SEPARATION
Volume I
BIOLOGICAL SEPARATIONS
Series Editor: Nicholas Catsimpoolas
Massachusetts Institute of Technology
Cambridge, Massachusetts
Methods of Protein Separation, Volume
Hdited by NicflOlas Catsimpoolas • 1975
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METHODSOF
PROTEIN
SEPARATION
Volume 1
Edited by
Nicholas Catsimpoolas
Massachusetts Institute of Technology
Cambridge, Massachusetts
Springer Science+ Business Media, LLC
Library of Congress Cataloging in Pu blication Data
Main entry under title:
Methods of protein separation.
(Biological separations)
Includes bibliographies and index.
1. Proteins. 2. Separation (Technology) 3. Biologica! chemistry. J.
Catsimpoolas, Nicholas.
QD431.M47 574.1'9245'028 75-17684
ISBN 978-1-4757-1259-9 ISBN 978-1-4757-1257-5 (eBook)
DOI 10.1007/978-1-4757-1257-5
© 197 5 Springer Science+Business Media New York
Originally published by Plenum Press, New York 1975
Softcover reprint of the hardcover I st edition 1 975
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227 West 17th Street, New York, N.Y.I0011
United Kingdom edition published by Plenum Press, London
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No part of this book may be reproduced, stored in a retrieval system, OI transmitted,
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CONTRIBUTORS
John R. Cann, Department 0/ Biophysics and Genetics, University 0/ Colorado
Medical Center, Denver, Colorado 80220
Nicholas Catsimpoolas, Biophysics Laboratory, Department 0/ Nutrition and
Food Science, Massachusetts Institute 0/ Technology, Cambridge,
Massachusetts 02139
Alfred J. Crowle, Webb-Waring Lung Institute and Department 0/ Micro
biology, University 0/ Colorado, School 0/ Medicine, Denver, Colorado
80220
Pedro Cuatrecasas, Department 0/ Experimental Therapeutics and Medicine,
The lohns Hopkins University School 0/ Medicine, Baltimore, Maryland
21205
James W. Drysdale, Department 0/ Biochemistry and Pharmacology, Tu/ts
University School 0/ Medicine, Boston, Massachusetts 02111
Robert E. Feeney, Department 0/ Food Science and Technology, University 0/
California, Davis, California 95616
Eli Grushka, Department 0/ Chemistry, State University 0/ New York at
Buffalo, Buffalo, New York 14214
James B. 11ft, Department 0/ Chemistry, University 0/ Redlands, Redlands,
California 92373
David T. Osuga, Department 0/ Food Science and Technology, University 0/
California, Davis, California 95616
Indu Parikh, Department 0/ Pharmacology, The lohns Hopkins University
School 0/ Medicine, Baltimore, Maryland 21205
Burton A. Zabin, Bio-Rad Laboratories, Richmond, California 94804
,.
PREFACE
This open-end treatise on methods concerning protein separation had its
beginning in an American Chemical Society symposium entitled "Con
temporary Protein Separation Methods" which was held in Atlantic City,
New Jersey in September 1974. The purpose of the symposium-and subse
quently of the present work-was to review the available modern techniques
and underlying principles for achieving one of the very important tasks of
experimental biology, namely the separation and characterization of proteins
present in complex biological mixtures. Physicochemical characterization
was covered only as related to the parent method of fractionation and there
fore involved mostly mass transport processes. Additionally, the presentation
of methods for gaini.ng insight into complex interacting protein profiles was
considered of paramount importance in the interpretation of separation
patterns. Finally, specific categories of proteins (e.g., chemically modified,
deriving from a specific tissue, conjugated to different moieties, etc.) require
meticulous trial and selection andjor modification of existing methodology
to carry out the desired separation. In such cases, the gained experience
provides valuable guidelines for further experimentation.
Although powerful techniques exist today for the separation and related
physicochemical characterization of proteins, many biological fractionation
problems require further innovations. It is hoped that the description in the
present treatise of some of the available separation tools and their limitations
will provide the necessary integrated background for new developments in
this area.
Nicholas Catsimpoolas
Cambridge, Massachusetts
vii
CONTENTS
Chapter 1
Sedimentation and Gel-Permeation Chromatography of
Associating-Dissociating Macromolecules:
The Role of Ligand Mediation and Rates of Reaction
lohn R. Cann
1. Introduction 1
11. Theoretical Formulation 2
111. Results 7
A. Ligand-Mediated Association-Dissociation Reactions 8
B. Kinetically Controlled Nonmediated Interactions 17
IV. Discussion 23
V. References 25
Chapter 2
Trans Electrophoresis
Nicholas Catsimpoolas
1. Introduction 27
A. Principle 27
B. History of in Situ Electro-Optical Scanning 28
C. Types of Electrophoresis and Supporting Media 29
D. Anatomy of the TRANS Electrophoresis System 33
11. Instrumental Aspects 34
A. The Electro-Optical Unit 34
B. The Scanning Stage Module 36
C. The Scanner Control Servo Unit 36
D. The Electrophoresis Cell Cassette . 37
E. The FillingjPurgingjCooling Module 38
ix
x CONTENTS
F. Other Components . 39
G. The Digital Data Acquisition Module 39
H. General Operational Procedure 39
I. Column-Coating Procedure 40
In. Data Processing 40
A. Slope Analysis . 40
B. Moment Analysis 43
IV. TRANS-CZE and TRANS-MZE 47
A. Preliminary Considerations 47
B. Peak Velocity 47
C. Kinetic Peak-Variance Measurements 50
D. Resolution. 51
E. Boundary Displacement . 51
V. TRANS-IF 52
A. Preliminary Considerations 52
B. Minimal Focusing Time. 53
C. Segmental pH Gradient and Apparent Isoelectric Point 57
D. Resolving Power and Resolution . 58
E. Retardation Coefficient . 59
F. Kinetics of Defocusing and Refocusing 60
G. Nonideal Effects in TRANS-IF 63
VI. Future Developments 65
VII. References 66
Chapter 3
Immunodiffusion
Alfred J. Crowle
I. Introduction 69
I I. Antigen - Antibody Reactions 69
IIl. Immunodiffusion 73
IV. Immunodiffusion Combined with Electrophoresis 82
V. HelpfuI Hints 89
VI. References 91
Chapter 4
Isoelectric Focusing in Poly acryl amide Gel
farnes W. Drysdale
I. Introduction 93
CONTENTS xi
11. Background 94
III. Methodology 99
A. Apparatus 99
B. Electrolyte Solutions 101
C. Gel Composition 102
D. Electrolysis Conditions 105
E. SampIe Application . 106
F. SampIe Load 107
G. Measurement of pR Gradients 107
R. pR-Gradient Instability 108
IV. SampIe Detection 109
A. Staining for Proteins 109
B. Ristochemical Staining 110
C. Gel Fractionation 113
D. Recovery of Focused Zones 113
E. Markers 114
V. Applications 114
A. Ferritins 114
B. Remoglobins 117
C. Interacting Systems 119
D. Nucleic Acids 121
E. Two-Dimensional Procedures 122
VI. Discussion 123
VII. References 125
Chapter 5
Purification of Chemically Modified Proteins
Robert E. Feeney and David T. Osuga
1. Introduction 127
11. General Types and Purposes of Chemical Modification 128
A. Modification to Change Biological Activity 128
B. Modification to Change Physical Properties 130
C. Modifications to Block Deteriorative Reactions . 131
D. Introduction of a Label . 133
E. Reversible Modifications 133
III. Some Problems Encountered in Chemical Modification 134
A. Denaturation 134
B. Reversibility of Modification . 135
xii CONTENTS
C. Heterogeneity of Products 136
D. A Product with Properties Very SimiIar to Those of the
Original Native Protein . 138
E. Side Reactions of a Chemical Nature Occurring During
Modification 138
IV. Analysis of Chemically Modified Proteins . 139
A. Analysis of Amino Acid Residues 140
V. Typical Examples of the Purification of Chemically Modified
Proteins 146
A. Heterogeneity Commonly Encountered in the Introduction
of Groups Causing Changes in Charges 146
B. Modifications with No Change in Charge . 147
C. So me Special Procedures for Chromatography 148
VI. The Use of Chemical Modification as a Tool for Purification
of Proteins 151
A. Purification by Reversible-Complex Formation 153
B. Reversible Chemical Modification for Separating Hybrids
of Variants of Oligomeric Proteins 154
C. The Use of Chemical Modification to Separate Products
with Different Biochemical Activities . 155
D. Reversible Modification to Allow for Another Modification 157
E. The Use of Cross-Linking Agents to Determine Degree of
Polymerization and Localization of Proteins in Tissues 159
VII. References 159
Chapter 6
Chromatographie Peak Shape Analysis
EU Grushka
I. Introduction 161
11. Moment Analysis 162
A. Definition 162
B. Mass Balance and the Moments 164
C. Other Uses of Moments Analysis 171
IU. Experimental Studies 176
A. Verification of Skew and Excess Utility 176
B. Peak Identification 179
IV. Slope Analysis 182
V. Conclusion 190
VI. References 190